Karina Kwok, Ph.D.
Associate Principal Scientist
MPI Research
Can you walk us through the basics of assessing the homogeneity of a dose formulation?
Homogeneity of a preclinical dose formulation refers to the uniform distribution of a test article in a vehicle, whether suspended in a liquid vehicle (suspension), dissolved in a liquid vehicle (solution), or mixed as a solid into a solid, dietary-type vehicle (solid). Assessment of homogeneity is a requirement referred to in the GLP regulations 21CFR Part 58, Section 58.113 Mixtures of articles with carriers.
The general approach for homogeneity assessment is to take samples from different layers of a prepared batch and analyze them for concentration. For a suspension, samples are taken from the top, middle, and bottom layer during stirring. At MPI Research, our approach for this method is to take six samples from each layer; two samples from each layer are analyzed for concentration/homogeneity assessment, while the remaining layers are kept for backup.
The typical acceptance criteria for a suspension-type preclinical dose formulation are as follows: an average percent recovery of 100±15% of the nominal (prepared) concentration, and a precision of ≤10% RSD for all samples analyzed. If the acceptance criteria are met, the prepared dose formulation is then considered to be homogeneous. For solution formulations, homogeneity assessment is to ensure the test article is uniformly dissolved in the liquid vehicle without any concentration gradients. Typical acceptance criteria for a solution-type preclinical dose formulation are: an average percent recovery of 100±10% of the nominal (prepared) concentration, and a precision of ≤5% RSD for all samples analyzed. To evaluate the homogeneity of a solid formulation, samples are taken from different strata of the formulation in the mixing vessel after the mixing steps have been completed. The typical acceptance criteria for a solid-type preclinical dose formulation analyzed using an HPLC-UV method are: an average percent recovery of 100±20% of the nominal (prepared) concentration, and a precision of ≤15% RSD for all samples analyzed.
What analytical methodology is most common for assessment?
Basically, the analytical methodology used for homogeneity assessment is a method for concentration determination. The following factors should be considered:
- The analyte and its physicochemical characteristics
- The vehicle and the components used for its preparation
- The dose concentration ranges to be measured
For example, for a suspension formulation that involves a small molecule test article with a chromophore in a vehicle of 0.5% methylcellulose in water, a reversed-phase HPLC method (RP-HPLC) with UV detection is a suitable choice for dose concentration measurements.
For proteins in a solution formulation in saline, total protein concentration determination using a UV method (A280) is a suitable approach.
For a solid formulation, depending on the complexity of the solid vehicle matrix and the physicochemical characteristics of the test article, either a RP-HPLC method with UV detection or an LC/MS method is suitable.
Factors, such as mixing methods or batch size can affect the homogeneity of dose formulations. What are some additional factors that can affect homogeneity?
The particle size of the test article can affect the homogeneity of suspensions. MPI Research has found that test articles that are of a finer and more uniform particle size are more uniformly distributed in a suspension. To assist in particle size reduction, homogenization and/ or sieving could be performed as part of the formulation preparation. Another factor affecting homogeneity is the final viscosity of the formulation. Adjustment or change of vehicle components could provide a formulation of higher or lower viscosity, leading to more uniform mixing and distribution. In the case of viscosity reduction, a surfactant such as Tween 80 can be used.
Can you tell us about some of the challenges you encountered with homogeneity for a formulation, and the steps you have taken to solve the problem?
The majority of the homogeneity challenges encountered are related to suspension formulations.
Case study #1 - The test article was a small molecule, provided in a solid form. The vehicle was 0.5% carboxymethylcellulose in water and the dose formulation was a suspension. During preparation, it was noticed that particles of the test article floated on top of the vehicle. The resolution was to slowly add vehicle to the test article while grinding using a mortar and pestle. Vehicle was continually added to the mortar with continued grinding to form a less viscous mix that was transferred to a calibrated measuring device.
Case study #2 - The test article was provided in a large particle size which did not facilitate uniform distribution in a suspension. Homogenizers were used to reduce the particle size in the formulation. To avoid problems that may have been associated with high homogenization speeds, extended stirring was used to remove the air, as well as sonication.
A best practice to minimize potential homogeneity problems is to prepare a test batch. Obviously, the feasibility of a test batch will depend on the availability of test article, as more of it will be required in addition to that used in the preclinical study itself.
The use of test batches is common for solid and dietary (solid-in-solid) formulations. In these cases, grinding and/or sieving of the test article and increased blending time may be needed to achieve acceptable homogeneity. Another approach is to add an additional carrier to the mixture to aid in dispersion.